Midbrain dopaminergic (mDA) neurons critically control voluntary movement, reward, and mood-related behaviors, and their degeneration/dysfunction is associated with major brain disorders such as Parkinson's disease (PD) and schizophrenia. In-depth understanding of molecular pathways underlying mDA neuron development will be crucial for effective therapeutic approaches of these diseases. To investigate the developmental mechanisms of the DA and noradrenaline (NA) systems, we previously proposed to study the transcriptional regulation of two hallmark genes encoding tyrosine hydroxylase (TH) and dopamine [unreadable]-hydroxylase (DBH), enzymes that catalyze the conversion of tyrosine to L-dopa, and of DA to NA, respectively. We have delineated the transcriptional mechanisms of TH and DBH gene expression and identified several key transcription factors that critically control these neuronal developments. In particular, we found that functional knockout of the homeodomain transcription factor Pitx3 uniquely leads to specific degeneration of A9 DA neurons of the substantia nigra and their nigrostriatal pathway, mimicking the pathological PD condition. Based on these progresses, we will focus on the transcriptional regulatory cascades/pathways during mDA neuronal development by focusing on further elucidation of the mechanisms of action and regulation of Pitx3 during different stages of mDA neuron development. We will define their functional roles, identify downstream targets, investigate the mechanisms of action, and their potential roles for A9-specific vulnerability. In addition, we will identity upstream regulator(s) of Pitx3 gene expression and how these factors functionally interplay with each other during mDA development for regulating neurogenesis, precursor specification, and mDA terminal differentiation. Finally, using our DA-specific gene delivery method, we will test if gene therapy approach of these key factors may ameliorate behavioral symptoms in PD animal model(s). Our proposed experiments will not only shed further insights into the molecular pathways of mDA neuron development but also translate into the study of related disease mechanisms as well as open the door to novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: Midbrain dopamine (mDA) neurons play critical roles in the regulation of voluntary movement, emotion, and reward-related behavior;their degeneration and/or dysfunction are associated with major brain disorders such as Parkinson's disease (PD), schizophrenia, and drug addiction. Based on our previous results indicating the critical role of Pitx3 for A9 mDA neuron development/maintenance, the proposed studies are aimed at elucidating the regulatory cascades/networks of the transcription factor Pitx3 during different stages of mDA neuron development. These studies will not only identify the molecular pathways underlying specification and maintenance of these neurons but may also provide novel insights for disease mechanisms and development of effective therapeutic approaches for related brain disorders.